The fluid shear stress acting on the vascular endothelial cells (ECs) due to blood flow induces mechanotransduction to regulate vascular functions in health and disease. The pulsatile shear stress in the straight part of the arterial tree has a long-term effect of suppressing the signaling pathways for proliferation (e.g., ERK) and hence causes growth arrest. In contrast, the complex flow pattern in the branch points is associated with reciprocating shear stress and causes a sustained activation of the signaling pathways for proliferation. We propose that the temporal controls of mechanosensing and mechanotransduction play an important role in the shear-regulation of EC functions and that this involves the interplay of mechanosensensing mechanisms by a balance between phosphorylation and dephosphorylation. We hypothesize that fluid shear stress with a significant forward direction, e.g., steady or pulsatile laminar shear stress causes initial Flk1/SHP2 association and ERK phosphorylation, followed by AT2/SHP1 dissociation and ERK dephosphorylation, thus providing the molecular mechanism for the transient nature of the shear-activation of ERK. In contrast, reciprocating shear stress with very little net flow would not cause the AT2/SHP1 dissociation and ERK dephosphorylation, thus keeping the ERK activation at a sustained level, with the consequent enhancement of proliferation. We also hypothesize that such interactions of mechanosensors and the time-dependent modulation of ERK activation are mediated through cell surface lipid rafts and the interaction of SHPs with the scaffolding protein Gab. Our hypothesis will be tested under the following four Specific Aims: 1) To investigate the effects of different flow patterns on Flk1/SHP2 and AT2/SHP1 interactions in the cell membrane. 2) To elucidate the roles of the scaffold proteins Gab, Grb and Sos in the Flk1/SHP2 and AT2/SHP1 interactions in response to different patterns of flow. 3) To assess the roles of SHP1 and SHP2, through Src, in the temporal changes of ERK response to different patterns of flow. 4) To determine the roles of Flk1/SHP2 and AT2/SHP1 interactions in the modulation of EC proliferation by different patterns of flow. The results will enhance our knowledge on the networking of signaling events in regulating the on/off switchings of the cellular responses to mechanical stimuli and will lead to a better understanding of how cells adapt to their environment and maintain vascular homeostasis in health and disease.